February 2nd, 2011 - Keele, UK: RecipharmCobra Biologics, the specialist biologics process development and cGMP manufacturing division of the international contract development and manufacturing organisation, Recipharm AB, today announces that the European patents on its ‘Xer-cise’ genetic engineering technology have been granted. Xer-cise enables bacteria to be genetically modified without leaving antibiotic resistance genes on their chromosomes. Xer-cise therefore overcomes the problems caused by the biosafety risk of potentially spreading antibiotic resistance to pathogens, the limited number of available antibiotic resistance genes and the competing use of these genes on plasmids. Xer-cise utilises Xer recombinases, which are naturally present in virtually every bacterial species.

Genetic modification is achieved by inserting a DNA cassette containing an antibiotic resistance gene, so that modified bacteria can be identified by their ability to survive in the presence of the antibiotic. By placing the sites recognised by Xer recombinases (dif sites) either side of the antibiotic resistance gene, the recombinases excise the gene when cells are grown later with no antibiotic present. Xer-cise has been demonstrated in bacteria including Escherichia coli, Salmonella, Bacillus subtilis and Mycobacterium, is applicable to many other bacteria.

Simon Saxby, VP of Biologics at RecipharmCobra Biologics, said: “Xer-cise has enabled molecular biologists at RecipharmCobra and elsewhere to rapidly construct bacterial strains that are not antibiotic resistant, and are therefore advantageous for the commercial production and delivery of biologics. We anticipate that this technology will greatly simplify and accelerate the genetic modification of many species of bacteria.”

About bacterial genetic modification

When bacteria are genetically modified, an antibiotic resistance gene is inserted into the chromosome adjacent to the modified region to enable bacterial cells that have undergone the mutation to be identified, as these cells will form colonies on agar plates containing the antibiotic. This can result in cells that contain multiple antibiotic resistance genes that reduce their versatility for replicating plasmids, which are circular DNA molecules that also contain antibiotic resistance genes in addition to a gene that produces the required protein. Also, only a limited number of genetic modifications can be undertaken in the same cell due to the limited number of available antibiotic resistance genes. There is therefore a requirement to remove the chromosomally inserted antibiotic resistance gene. Existing strategies require the insertion of a second plasmid following the chromosomal modification. This plasmid expresses a recombinase (e.g. Cre or Flp) that recognises sites flanking the antibiotic resistance gene and excise it from the chromosome. The plasmid then needs to be removed from the cell. This process therefore introduces additional stages, and there are numerous species of bacteria for which a suitable recombinase plasmid has not been developed.

About Xer-cise™ Technology

The Xer-cise technology uses native Xer recombinases that normally function to restore the chromosomal and plasmidal dimers generated by the enzyme RecA back to monomers. These Xer recombinases are ubiquitous in bacteria. An antibiotic resistance gene is flanked by 28 base-pair long dif sites, which are in turn flanked by chromosomal target homology. This cassette is either constructed on a plasmid and linearised, or assembled by PCR, and transformed into the target bacterium. Homologous recombination carried out by other enzymes precisely inserts the gene cassette into the chromosome. Gene integration mutants are selected on agar plates containing the antibiotic. These are then cultured in antibiotic-free medium, and the Xer recombinases recombine the two dif sites to a single site, thereby excising the intervening antibiotic resistance gene to generate the new mutant strain.

About RecipharmCobra Biologics

RecipharmCobra Biologics has a proven track record in the development and manufacture of monoclonal antibodies, recombinant therapeutic proteins, viruses and DNA as well as cell based vaccines and therapies for clinical trials and regulatory approvals. RecipharmCobra Biologics has two GMP approved production facilities, from which a comprehensive service is provided; including cell line development, process and analytical development, GMP manufacture, coordination of formulation and fill/finish activities, and release to clinic. RecipharmCobra is the specialist biologics division of Recipharm AB.

www.recipharmcobrabio.com

About Recipharm

Recipharm AB is a leading contract development and manufacturing organisation based in Sweden with 2,000 employees. The company operates 12 development and manufacturing facilities in Sweden, France, the UK, Spain and Germany and is headquartered near Stockholm. We supply the global pharmaceuticals market with hundreds of different products in most dosage forms, including solid dose, granulates, powders, sterile liquids and lyophilisates, semisolids, beta-lactams, hormones, and inhalers.